Endothelial and epithelial cells are a major target in vivo for HCMV and potentially an important site for initiation of virus transfer across the placenta to the fetus in congenital infection. In HCMV, the gH/gL glycoprotein complex and viral proteins from the UL128-131 locus are required for endocytic viral entry into endothelial/epithelial cells. This viral entry pathway is separate from the gB entry pathway found in fibroblast cells. Antibodies against proteins of the UL128-UL131 locus and endocytic complex can neutralize virus infection of endothelial/epithelial cells. We hypothesize that a vaccine strategy which includes components of the endocytic viral entry pathway as neutralizing target antigens would be a more effective vaccine against congenital infection. The studies will be carried out using guinea pig cytomegalovirus (GPCMV) because of species specificity of HCMV. The guinea pig is the only small animal model that allows the study of congenital cytomegalovirus infection. An added complication of studying bona fide full length CMV and virus entry into endothelial/epithelial cells is the propensity for deletion of the UL128-131 locus when virus is grown on fibroblast cell lines. This happens in both HCMV and GPCMV, which complicates infection studies on endo/epithelial cells. We propose to use a novel approach of a lentivirus transduced tet-off complementing cell line system expressing missing GPCMV genes (GP128-131) to produce pseudotyped GPCMV encoding part or all of the components of the presumed endocytic pathway. Our hypothesis is that we will be able to determine the relevant proteins necessary for the endocytic pathway of GPCMV infection of endothelial cells via this strategy. Furthermore, we hypothesize that once the minimum components of the endocytic pathway protein complex are identified they can be included as part of a novel vaccine strategy based on a replication impaired (DISC) virus which will enhance the protective immune response against congenital CMV compared to a DISC virus lacking this locus. PUBLIC HEALTH RELEVANCE: Cytomegalovirus (CMV) is a ubiquitous pathogen that causes significant mortality and morbidity in immunocompromised populations including transplant and AIDS patients and the fetus in utero. Congenital CMV infection causes mental retardation and deafness in surviving newborn. CMV is the most common AIDS related secondary infection. There is no vaccine to CMV. Although current antivirals are available for transplant and AIDS patients these result in the emergence of resistant strains that cause disease. Additionally, antiviral drug toxic side effects preclude their use in the prevention of congenital CMV.